A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens
2.1. Water-Based Deparaffinization Competes with the Gold-Standard Xylene and Takes Only a Fraction of the Time
2.2. Efficient Tissue Homogenization Using Micropestles
2.3. Improved Protein Extraction with Sodium Deoxycholate (SDC)
2.4. PAC and STRAP Are Good Alternatives to FASP
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Source of Specimens
4.3. Sample Preparation of Core Needle Biopsy-Sized Specimens
4.3.1. Optimization of Deparaffinization
- depX : The samples were washed with 1 mL of 100% xylene and incubated for 10 min at room temperature (RT), followed by centrifugation at 14,000× g for 2 min and disposal of the supernatant, followed by another 2 repetitions. Then, the samples were washed twice each with 1 mL of 100%, 96%, and 70% ethanol, followed by incubation for 1 min at RT and centrifugation as above.
- depW (modified from ): The samples were washed 2× with 500 µL of hot deionized water and incubated for 1 min at RT under vigorous vortex mixing. Each washing step was followed by centrifugation at 20,000× g for 5 min at 4 °C. The supernatant, containing paraffin either floating on the liquid surface or stuck to the wall of the tube (Figure 7), was discarded and the deparaffinized and rehydrated core was transferred to a clean LoBind Eppendorf tube.
4.3.2. Optimization of Tissue Homogenization
4.3.3. Optimization of Protein Extraction
4.3.4. Optimization of Tryptic Digestion
- FASP was performed as described above.
- PAC was performed using amine microparticles (MagReSyn) based on Batth et al. . For 20 µg of protein lysate, 12 µL of microparticle stock solution (20 µg/mL) were equilibrated with 100 µL of 70% ACN, briefly vortexed and placed on a magnetic rack to remove the supernatant. This step was repeated another two times. Next, the protein extracts were added to the beads and the sample was adjusted to a final concentration of 70% ACN, thoroughly vortexed and incubated for 10 min at RT without shaking. The following washing steps were performed on a magnetic rack without disturbing the protein/bead aggregate. The supernatants were discarded, and the beads were washed on the magnetic rack with 1 mL of 95% ACN for 10 s, followed by a wash with 1 mL of 70% ACN without disturbing the protein/bead aggregate. The tubes were removed from the magnetic rack, 100 µL of digestion buffer (1:20 (w/w) trypsin:protein in 0.2 M GuHCl, 50 mM AmBic, 2 mM CaCl2) were added and the samples were incubated at 37 °C for 3 h. After acidification with trifluoroacetic acid (TFA) to a final concentration of 2%, the tubes were placed on the magnetic rack for 1 min, followed by removal of the supernatant. To remove residual beads, the samples were centrifuged at 20,000× g for 10 min. The supernatants were dried under vacuum and reconstituted in 0.1% FA for nano-LC-MS/MS.
- STRAP digestion was performed according to the manufacturer’s instructions . Lysate corresponding to 20 µg of total protein was acidified to a final concentration of 1.2% phosphoric acid. SDS was added to a final concentration of 2% followed by a 7-fold dilution with STRAP binding buffer (90% methanol, 100 mM Tris-HCl, pH 7.1). The sample was loaded onto the STRAP and centrifuged at 4000× g for 1 min, followed by three washes with 150 µL binding buffer, with the spin-column being rotated by 180° between centrifugation steps. Then, 200 µL of STRAP digestion buffer, comprised of 1:10 (w/w) trypsin:protein in 0.2 M GuHCl, 50 mM AmBic, 2 mM CaCl2 were added to the STRAP, which was briefly spun on a benchtop centrifuge to assure saturation of the column material with the digestion buffer. The flow-through was loaded again on top of the column. The sample was incubated at 47 °C for 3 h. Peptides were eluted by sequential elution (1000xg, 1 min) using 40 µL of 50 mM AmBic, 40 µL of 0.1% FA, and 35 µL of 50% ACN, 0.1% FA. The collected peptide sample was dried under vacuum and reconstituted in 0.1% FA for nano-LC-MS/MS.
4.4. Data Analysis
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Mitsa, G.; Guo, Q.; Goncalves, C.; Preston, S.E.J.; Lacasse, V.; Aguilar-Mahecha, A.; Benlimame, N.; Basik, M.; Spatz, A.; Batist, G.; et al. A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens. Int. J. Mol. Sci. 2022, 23, 4443. https://doi.org/10.3390/ijms23084443
Mitsa G, Guo Q, Goncalves C, Preston SEJ, Lacasse V, Aguilar-Mahecha A, Benlimame N, Basik M, Spatz A, Batist G, et al. A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens. International Journal of Molecular Sciences. 2022; 23(8):4443. https://doi.org/10.3390/ijms23084443Chicago/Turabian Style
Mitsa, Georgia, Qianyu Guo, Christophe Goncalves, Samuel E. J. Preston, Vincent Lacasse, Adriana Aguilar-Mahecha, Naciba Benlimame, Mark Basik, Alan Spatz, Gerald Batist, and et al. 2022. "A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens" International Journal of Molecular Sciences 23, no. 8: 4443. https://doi.org/10.3390/ijms23084443